Detection of BRAF V600E in cancers: Focus on BRAF V600E (VE1) Mouse Monoclonal Primary Antibody scientific overview

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Join us as we explore the BRAF V600E mutation, which accounts for approximately 80% of all BRAF mutations. We will describe the use of IHC to detect this mutation using an antibody specific for the BRAF V600E mutation. After this presentation, you will have a greater understanding of how the VENTANA BRAF V600E (VE1) Mouse Monoclonal Antibody helps you evaluate the presence of this mutation within the context of colorectal cancer, thyroid cancer and hairy cell leukemia.

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Detection of BRAF V600E in cancers: Focus on BRAF V600E (VE1) Mouse Monoclonal Primary Antibody scientific overview

  1. 1. Detection of BRAF V600E in cancers Focus on BRAF V600E (VE1) Mouse Monoclonal Primary Antibody Scientific Overview
  2. 2. Introduction BRAF V600E (VE1) Mouse Monoclonal Primary Antibody Colorectal cancer Thyroid cancer Hairy cell leukemia Conclusion 3
  3. 3. Introduction BRAF V600E (VE1) Mouse Monoclobal Primary Antibody Colorectal cancer Thyroid cancer Hairy cell leukemia Conclusion 4
  4. 4. BRAF is a serine threonine kinase v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) • Part of the RAS/MAPK signaling pathway • Drives cell proliferation, survival, differentiation1 1.  Wan et al. Cell, Vol. 116, 855–867, March 19, 2004. Roring M, et al. The EMBO Journal (2012) 31, 2629–2647. Davies H, et al. NATURE |VOL 417 | 27 JUNE 2002 5
  5. 5. BRAF V600E is the most common mutant >30 different BRAF mutations have been described, however, “…the T1799A transversion mutation in BRAF accounts for more than 80% of all known BRAF mutations. The protein product resulting from this mutation is a glutamic acid for valine substitution at codon 600 (V600E)….”2 2. Pakneshan S, et al. Pathology (June 2013) 45(4), pp. 346–356. 6
  6. 6. The BRAF V600E mutant is found in many cancers BRAF V600E mutations have been detected in –  Colorectal cancer –  Papillary thyroid carcinoma –  Melanoma –  Hairy cell leukemia –  Primary central nervous system cancers –  Lung cancers 7
  7. 7. Distribution of BRAF mutations by cancer Percent (%) with BRAF mutations Percent (%) that are BRAF V600E mutations Colorectal 5-152 >904 Papillary thyroid 40-703 >985 Hairy cell leukemia ~1003 ~1003 ~40-603 70-903 Cancer Melanoma* *Content should be used only for scientific discussions only. The BRAF IHC should not be promoted in Melanoma 2 Pakneshan S,, et al. Clinicopathological relevance of BRAF mutations in human cancer. Pathology. 2013 Jun; 45(5): 346-356. 3. Capper D, Preusser M, et al. Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody. Acta Neuropathol. 2011 Jul; 4. Sharma S. BRAF Mutation Testing in Colorectal Cancer. Arch Pathol Lab Med. 2010; 134: 1225-1228. 5. Bullock M, O’Neill C, Chou A, et al. Utilization of a MAB for BRAF V600E detection in papillary thyroid carcinoma. Endocrine-Related Cancer. 2012; 19: 779-784. 8
  8. 8. BRAF V600E mutant is constitutively active In addition, BRAF V600E accounts for >90% of activating mutations6 “The protein product resulting from this mutation is a glutamic acid for valine substitution at codon 600 (V600E), leading to hyperactivation of the MAPK pathway. The result of this deregulated downstream signaling is unregulated cell proliferation and survival, factors that contribute to oncogenesis.”4 Colorectal cancer positive for BRAF V600E Detected by VE1 with OptiView DAB IHC detection (20x) 4. Sharma S, et al. Arch Pathol Lab Med. 2010;134:1225–1228 6. Pakneshan S, et al. Pathology (June 2013) 45(4), pp. 346–356. 9
  9. 9. Introduction BRAF V600E (VE1) Mouse Monoclonal Primary Antibody Colorectal cancer Thyroid cancer Hairy cell leukemia Conclusion 10
  10. 10. BRAF V600E (VE1) mutation specific antibody •  Mouse monoclonal antibody3,7 •  Specific for BRAF V600E mutant protein3,7 •  The staining pattern for anti-BRAF V600E (VE1) antibody is cytoplasmic staining of tumor cells. •  Evaluate for the presence of this mutant with tissue context3,7 •  Easily integrated into the anatomic pathology laboratory 3. Capper D et al. Acta Neuropathol (2011) 122:11–19. 7.Capper D, et al. Oncotarget 2012; 3: 907-908. Capper D, et al. Int J Cancer. 2013 Mar 30. doi: 10.1002/ijc.28183. 11
  11. 11. BRAF V600E (VE1) mutation specific antibody •  High concordance with Sanger sequencing >95%3,7 •  Can detect mutant protein when low mutant levels limits molecular approaches (minimum ~25% of mutant allele) 3,7 •  Does not require microdissection3,7 3. Capper D et al. Acta Neuropathol (2011) 122:11–19. 7. Capper D, et al. Oncotarget 2012; 3: 907-908. Capper D, et al. Int J Cancer. 2013 Mar 30. doi: 10.1002/ijc.28183. 12
  12. 12. BRAF V600E (VE1) mutation specific antibody VE1 IHC compared to Sanger sequencing in MSI-H colorectal cancers demonstrated Sensitivity 100% Specificity 98.8% 8 8. Capper D, et al. Int J Cancer. 2013 Mar 30. doi: 10.1002/ijc.28183 13
  13. 13. Colorectal cancer positive for BRAF V600E. Detected by VE1 with OptiView DAB IHC detection (20x) Ventana Medical Systems, Inc. 14
  14. 14. Thyroid cancer positive for BRAF V600E. Detected by VE1 with OptiView DAB IHC detection (20x) Ventana Medical Systems, Inc. 15
  15. 15. BRAF V600E (VE1) Mouse Monoclonal Primary Antibody Package insert highlights Intended use statement Anti-BRAF V600E (VE1) Mouse Monoclonal Primary Antibody may be used to aid in the identification of the mutant protein, BRAF V600E. The antibody is intended for qualitative staining in sections of formalinfixed, paraffin-embedded tissue. This product should be interpreted by a qualified pathologist in conjunction with histological examination, relevant clinical information and proper controls. This antibody is intended for in vitro diagnostic (IVD) use. Summary and explanation The BRAF gene located on chromosome 7q34 encodes a cytoplasmic serine-threonine kinase that acts downstream of the mitogen-activated protein kinase (MAPK) signaling pathway. Oncogenic mutations in BRAF gene, all within the kinase domain, constitutively activate MAPK signaling pathway resulting in increased cell proliferation and apoptosis resistance. The most common of all activating BRAF mutations (T1799A point mutation) results in a substitution of valine (V) to glutamic acid (E) at the position 600 of the amino acid sequence. BRAF V600E mutations were detected in approximately 8% of all solid tumors, including 43% of melanomas, 39% of papillary thyroid carcinomas, 12% of serous ovarian carcinomas, 12% of colorectal adenocarcinomas, 2% of lung cancers and other cancers. Furthermore, the BRAF V600E mutation has been recently described as a molecular marker of hairy cell leukemia. The anti-BRAF V600 (VE1) antibody is a mouse monoclonal antibody (clone VE1) produced against synthetic peptide representing the BRAF mutated amino acid sequence from amino acid 596 to 606 (GLATEKSRWSG). This mutation-specific antibody exhibits a cytoplasmic staining pattern. This antibody differentiates V600E mutation in the BRAF protein from the wild type BRAF protein and the other BRAF mutated proteins. 16 16
  16. 16. BRAF V600E (VE1) Mouse Monoclonal Primary Antibody Package insert highlights – Specific limitations Specific Limitations “The user must validate individual laboratory optimized results obtained with this reagent. This assay was optimized with OptiView DAB IHC Detection Kit and it is not recommended to be used with ultraView Universal DAB Detection Kit. The specimen should be fixed within 2 hours after collection for at least 12 hours with 10% neutral buffered formalin. It is not recommended to fix tissues with 95% alcohol, Prefer fixative, Z-5 fixative or alcohol formalin acetic acid (AFA). Anti-BRAF antibody (VE1) was found to occasionally exhibit cytoplasmic background staining in smooth muscle and nuclear staining in normal colon epithelial cells, enterocytes, Leydig cells of testis, adrenal gland, pituitary gland and some tumor cells; however, such cases should not be considered as positive for the BRAF V600E mutation” Confidential and proprietary to Ventana Medical Systems, Inc. For internal use only. Do not copy. Do not distribute. 17
  17. 17. BRAF V600E (VE1) Mouse Monoclonal Primary Antibody Package insert highlights… cont. Staining interpretation “The staining pattern for anti-BRAF V600E (VE1) antibody is cytoplasmic staining of tumor cells. Positive cases must show cytoplasmic staining in tumor cells when the anti-BRAF V600E (VE1)…. Nuclear staining in tumor cells is sometimes observed” Nuclear staining Cytoplasmic staining Positive colorectal cancer for BRAF V600E with (cytoplasmic staining) Nuclear staining Negative colorectal case for BRAF V600E with 18 Nuclear staining 18
  18. 18. Introduction BRAF V600E (VE1) Mouse Monoclobal Primary Antibody Colorectal cancer Thyroid cancer Hairy cell leukemia Conclusion 19
  19. 19. Colorectal cancer: key facts Third most common cancer globally11 Fourth leading cause of cancer death11 Geography Age group Incidence number Worldwide All 1,233,711 Incidence rate Population (per 100,000) 17.3 7,095,217,980 12,13 11. Haggar F, et al. CLINICS IN COLON AND RECTAL SURGERY/VOLUME 22, NUMBER 4 2009. 12. GLOBOCAN 2008. Cancer Fact Sheet. Colorectal. American Cancer Society. http://www.cancer.org/cancer/colonandrectumcancer/detailedguide/colorectal-cancer-key-statistics. 13. American Cancer Society http://www.cancer.org/cancer/colonandrectumcancer/detailedguide/colorectal-cancer-survival-rates 20
  20. 20. Colorectal cancer: key facts Sporadic vs. Inherited - Vast majority attributed to sporadic versus inherited15 15. Aaltonen et al. Clin Cancer Res. 2007: 356-361. 21
  21. 21. Colorectal cancer: key facts Microsatellite status -  Most are Microsatellite stable (MSS) -  A subset demonstrate microsatellite instabilityhigh (MSI-H) 8 8. Capper et al. 2013. Int J cancer 22
  22. 22. BRAF V600E in colorectal cancer, Kalady et al.: Associated with MSI Retrospective analysis at the Cleveland Clinic, N = 475 CRC BRAF V600E detected by PCR BRAF mutations in 56 (12%) BRAF V600E comprised 55 (98%) of detected mutations BRAF V600E vs. wildtype, p value <0.001 BRAF V600E in 76% of MSI cases BRAF wildtype in 16% of MSI cases17 17. Kalady MF., et al.Dis Colon Rectum 2012; 55: 128–133. 23
  23. 23. BRAF V600E in colorectal cancer, Jensen et al.: Associated with MLH1 deficiency Successive CRC patients N=262 evaluated by MMR IHC MMR deficient n =39, 14.9% (36 MLH1, 3 MSH2) MLH1 deficient n =36 (92%) BRAF V600E by direct sequencing 32 (89%) 10 10. Jensen LH, et al. 2007 The Association of Coloproctology of Great Britain and Ireland. Colorectal Disease, 10, 490–497. 24
  24. 24. BRAF V600E (VE1) in colorectal cancer: Desai et al. Sensitivity, Specificity, NPV, PPV19 19. Desai J, et al. ESMO 2012 Poster. Absract No.XXX 25
  25. 25. BRAF V600E (VE1) in colorectal cancer: Desai et al. Sensitivity, Specificity, NPV, PPV19 19. Desai J, et al. ESMO 2012 Poster. Absract No.XXX 26
  26. 26. BRAF V600E (VE1) in colorectal cancer: Desai et al. Sensitivity, Specificity, NPV, PPV 19. Desai J, et al. ESMO 2012 Poster. Absract No.XXX 27
  27. 27. Detection of BRAF V600E in CRC: VE1 IHC versus Sanger Sequencing VE1 demonstrated a concordance of 99% with sequencing (N=91) 8 VE1 IHC METHOD Sanger sequencing Positive Positive 11 Negative 1 Total 12 Negative Total 0 12 79 79 80 91 8. Capper 2013. Int J cancer . 28
  28. 28. Detection of BRAF V600E: Comparison of IHC and PCR in CRC resections VE1 IHC demonstrated a concordance of 99% with PCR (N=75) 9 METHOD PCR VE1 IHC Positive Positive 49 Negative 0 Total 49 Negative Total 1 50 25 25 26 75 9. Sinicrope et al. Cancer Month 00, 2013. 29
  29. 29. Introduction BRAF V600E (VE1) Mouse Monoclonal Primary Antibody Colorectal cancer Papillary thyroid cancer Hairy cell leukemia Conclusion 30
  30. 30. Thyroid cancer: key facts Worldwide incidence (2008) 212,033 cases 3.1/100,000 ASR Gender disparity Female predominance ~73% cases Males ~27% cases Papillary thyroid carcinoma Most common thyroid cancer20,21 20. Nikiforov 2013 USCAP Presentation. 21. Nikiforov YE, et al. J Clin Endocrinol Metab 94: 2092–2098, 2009. Incidence and Prevalence Database. http://www.tdrdata.com/default.aspx 31
  31. 31. Papillary thyroid cancer: key facts Thyroid nodules are common….cancer is rare “…among surgically removed thyroid nodules only 8% to 56% are found to be malignant.”21 21. Nikiforov YE, et al. J Clin Endocrinol Metab 94: 2092–2098, 2009 32
  32. 32. Papillary thyroid cancer: key facts About 10%-40% of thyroid nodule FNAs are indeterminate On diagnostic lobectomy for indeterminate FNAs – 75% are benign – 25% cancer •  These patients will need additional treatment: often total thyroidectomy and radioiodine therapy20,21 20. Nikiforov 2013 USCAP Presentation. 21. Nikiforov YE, et al. J Clin Endocrinol Metab 94: 2092–2098, 2009 33
  33. 33. Papillary thyroid cancer: key facts For those with an indeterminate FNA20,21 75% over-treated 25% under-treated 20. Nikiforov 2013 USCAP Presentation. 21. Nikiforov YE, et al. J Clin Endocrinol Metab 94: 2092–2098, 2009 34
  34. 34. BRAF V600E (VE1) in papillary thyroid cancer VE1 IHC versus direct DNA sequencing, N =144 Demonstrated 100% concordance22 BRAF V600E Status 22. Koperek O, et al. Am J Surg Pathol 2012;36:844–850) 35
  35. 35. BRAF V600E (VE1) in papillary thyroid cancer: Bullock et al. Compared VE1 IHC and Sanger sequencing N=101 well characterized papillary thyroid carcinomas 96 had molecular data, 10 discordant samples5 BRAF V600E status IHC n (%) Sanger Sequencing n (%) Positive 68 (71) 59 (61) Negative 28 (29) 37 (39) Total 96 (100) 96 (100) 5. Bullock M. Endocrine-Related Cancer (2012) 19 779–784 36
  36. 36. BRAF V600E (VE1) in papillary thyroid cancer Discordant samples were reevaluated by VE1 IHC, Sanger sequencing and massive parallel sequencing on macrodissected samples. VE1 IHC initially identified 7 more cases (17%) compared to Sanger sequencing5 BRAF V600E status IHC n (%) Sequencing n (%) Positive 10 (100) 7 (70) Negative 0 (0) 3 (30) Total 10(100) 10(100) 5. Bullock M. Endocrine-Related Cancer (2012) 19 779–784 37
  37. 37. Introduction BRAF V600E (VE1) Mouse Monoclonal Antibody Colorectal cancer Thyroid cancer Hairy cell leukemia Conclusion 38
  38. 38. Hairy cell leukemia: key facts A mature B-cell neoplasm Unlike most hematological cancers, no recurrent characteristic chromosomal abnormalities have been identified23 23. Andrulis M, et al. J Surg Pathol 2012;36:1796–1800) 39
  39. 39. BRAF V600E in hairy cell leukemia, Tiacci et al. Detected by Sanger sequencing Hairy cell leukemia BRAF V600E positive in 100% (48/48) Other peripheral B-cell lymphomas (including HCL variant, SMZL) BRAF V600E positive in 0% (0/195)24 24. Tiacci e, et al. N Engl J Med 2011;364:2305-15. 40
  40. 40. BRAF V600E in hairy cell leukemia, Arcaini et al. Allele specific PCR in 240 cases including 62 HCL Hairy cell leukemia 100% (62/62) BRAF V600E positive Other B cell chronic lymphoproliferative disorders 1 % (2/178) BRAF V600E positive25 25. Arcainina L et al. BLOOD, 5 JANUARY 2012 VOLUME 119, NUMBER 1. 41
  41. 41. Introduction BRAF V600E (VE1) Mouse Monoclonal Antibody Colorectal cancer Thyroid cancer Hairy cell leukemia Conclusion 42
  42. 42. VENTANA Empowering | Innovation www.roche.com www.ventana.com © 2014 Ventana Medical Systems, Inc. VENTANA, the VENTANA logo, OPTIVIEW and ultraView are trademarks of Roche. All other trademarks are the property of their respective owners. N4960-1 0214B
  43. 43. Doing now what patients need next

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